Demonstrating Interactions of Transcription Factors with DNA by Electrophoretic Mobility Shift Assay

2017 ◽  
pp. 11-21 ◽  
Author(s):  
Nasim Yousaf ◽  
David Gould
Keyword(s):  
Transcription Factors ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Mobility Shift ◽  
Mobility Shift Assay

scholarly journals Functions of PIT1 in GATA2-dependent transactivation of the thyrotropin β promoter

2008 ◽  
Vol 42 (3) ◽  
pp. 225-237 ◽  
Author(s):  
Yumiko Kashiwabara ◽  
Shigekazu Sasaki ◽  
Akio Matsushita ◽  
Koji Nagayama ◽  
Kenji Ohba ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Minimal Sequence ◽  
Mobility Shift Assay ◽  
Β Chain

Thyrotropin (TSH) is a heterodimer consisting of α and β chains, and the β chain (TSHβ) is specific to TSH. The coexistence of two transcription factors, PIT1 and GATA2, is known to be essential for TSHβ expression. Using kidney-derived CV1 cells, we investigated the role of PIT1 in the expression of Tshb gene. GATA2 Zn finger domain, which is known to recognize GATA-responsive elements (GATA-REs), is essential for cooperation by PIT1. Transactivation of TSHβ promoter requires PIT1-binding site upstream to GATA-REs (PIT1-US), and the spacing between PIT1-US and GATA-REs strictly determines the cooperation between PIT1 and GATA2. Moreover, truncation of the sequence downstream to GATA-REs enabled GATA2 to transactivate the TSHβ promoter without PIT1. The deleted region (nt −82/−52) designated as a suppressor region (SR) was considered to inhibit transactivation by GATA2. The cooperation of PIT1 with GATA2 was not conventional synergism but rather counteracted SR-induced suppression (derepression). The minimal sequence for SR was mapped to the 9 bp sequence downstream to GATA-REs. Electrophoretic mobility shift assay suggested that some nuclear factor exists in CV1 cells, which binds with SR and this interaction was blocked by recombinant PIT1. Our study indicates that major activator for the TSHβ promoter is GATA2 and that PIT1 protects the function of GATA2 from the inhibition by SR-binding protein.

scholarly journals From an electrophoretic mobility shift assay to isolated transcription factors: a fast genomic-proteomic approach

BMC Genomics ◽  
2010 ◽  
Vol 11 (1) ◽  
Author(s):  
Astrid R Mach-Aigner ◽  
Karin Grosstessner-Hain ◽  
Marcio J Poças-Fonseca ◽  
Karl Mechtler ◽  
Robert L Mach
Keyword(s):  
Transcription Factors ◽  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Mobility Shift ◽  
Proteomic Approach ◽  
Mobility Shift Assay

A fluorescence based non-radioactive electrophoretic mobility shift assay

2000 ◽  
Vol 78 (2) ◽  
pp. 163-170 ◽  
Author(s):  
K Ruscher ◽  
M Reuter ◽  
D Kupper ◽  
G Trendelenburg ◽  
U Dirnagl ◽  
...  
Keyword(s):  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Mobility Shift ◽  
Mobility Shift Assay

scholarly journals Human replication-dependent histone H3 genes are activated by a tandemly arranged pair of two CCAAT boxes

2004 ◽  
Vol 384 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Heiner KOESSLER ◽  
Joerg KAHLE ◽  
Christa BODE ◽  
Detlef DOENECKE ◽  
Werner ALBIG
Keyword(s):  
Electrophoretic Mobility Shift Assay ◽  
Electrophoretic Mobility ◽  
Histone H3 ◽  
Maximal Activity ◽  
Significant Loss ◽  
Tata Box ◽  
Mobility Shift ◽  
Nuclear Factor Y ◽  
Binding Factor ◽  
Mobility Shift Assay

We have analysed the transcriptional regulation of the human histone H3 genes using promoter deletion series, scanning mutagenesis, specific mutagenesis and electrophoretic mobility-shift assay experiments. The promoters of five of the six examined histone H3 genes showed near-maximal activity at lengths of 133–227 bp: H3/d 198 bp, H3/h 147 bp, H3/k 133 bp, H3/m 227 bp, H3/n 140 bp (exception H3/i). To search for functional cis-elements within these regions, we performed scanning mutagenesis of the two histone H3 promoters H3/k and H3/m. Mutagenesis revealed that the functional framework of the histone H3 promoters consists of a TATA box and two tandemly arranged CCAAT boxes in relatively fixed positions. Alterations of the distance between the CCAAT boxes and of the distance between the CCAAT boxes and the TATA box resulted in significant loss of activity. In electrophoretic mobility-shift assay experiments, the factor CBF (CCAAT-binding factor)/NF-Y (nuclear factor-Y) bound to isolated CCAAT boxes of the H3/k promoter. This suggests that an initiation complex is formed on the histone H3 promoter that has a defined structure and limited flexibility, consisting of two molecules of CBF/NF-Y and further (general or specific) transcription factors.

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